ELISA-based Zap70 competition assay

Immulon 4HBX microtiter plates were coated overnight with 50 ng/ml GST-phosphorylated CD3γ^CD plus 400 ng/ml GST in 50 mM Na₂CO₃, pH 9.2. Wells were washed with TBST to remove unadsorbed protein and then blocked for 1 h at room temperature with TBST plus 1% BSA. Competitor proteins were mixed, at the ratios indicated in Figure 5, with 5 μg/ml Zap70 D461N-tagged in exchange buffer (50 mM HEPES, pH 7.4, 140 mM KCl, 10 mM NaCl, 5 mM MgCl₂, 1% glycerol, 0.2% NP-40, 1 mM DTT and 0.5% BSA with or without 1 mM ATP). Protein mixtures were then added to the blocked wells and allowed to bind for 1 h at room temperature. Unbound protein was removed by washing with exchange buffer. Bound Zap70 D461N-tagged was detected with 1 μg/ml antibody to the Myc tag (clone 9E10, Fisher Scientific) or antibody to the HA tag (clone 12CA5, Becton Dickinson). Primary antibodies (Supplementary Table 7) were incubated for 1 h at room temperature and then washed with exchange buffer to remove unbound antibody. Primary antibodies were detected by incubation for 30 min at room temperature with 4 μg/ml secondary HRP-labeled anti-mouse (ThermoFisher #32430) in exchange buffer. Wells were washed, and then bound detection antibody was visualized colorimetrically with Fast-OPD (Sigma); color development was stopped with 3 N HCl according to the manufacturer's instructions. Absorbance at 490 nm was determined on a VersaMax microplate reader (Molecular Devices).